Arginine-free TNFR:Fc-fusion polypeptide compositions and methods of use

ABSTRACT

Aspects of the invention are directed to arginine-free polypeptide-containing compositions and methods for treating disorders associated with inflammation or the autoimmune response. In particular, the polypeptide is etanercept.

RELATED APPLICATIONS

This application is a National Phase Application under 35 U.S.C. § 371of International Application No. PCT/US2012/044988, filed Jun. 29, 2012,which claims the benefit under 35 U.S.C. § 119(e) of U.S. provisionalapplication No. 61/504,110, filed Jul. 1, 2011, the content of which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

Certain aspects of the invention relates to therapeuticpolypeptide-based compositions.

BACKGROUND

Therapeutic polypeptide preparations are often stored prior to use.Polypeptides, however, are unstable if stored in an aqueous form forextended periods of time, particularly in the absence of a stabilizingagent such as arginine. An alternative to relying on aqueous storage isto prepare a dry lyophilized form of a polypeptide, although,reconstitution of a dried polypeptide often results in aggregation ordenaturation.

SUMMARY OF THE INVENTION

In some embodiments, arginine-free polypeptide compositions areprovided. Formulations have been identified for preparing arginine-freepolypeptide solutions that are stable for an extended period of time.These formulations have several benefits relative to arginine-stabilizedsolutions, including reduced cost and a reduced incidence ofside-effects associated with the presence of arginine. Surprisingly, anaqueous polypeptide preparation can be stabilized by using a relativelyhigh salt concentration in the absence of arginine or other stabilizingamino acid (e.g., lysine or glycine or other stabilizing amino acid, forexample, one having a positive charge).

In some embodiments, provided herein are compositions comprising (orconsisting, or consisting essentially of): an isolated polypeptide(e.g., a therapeutic polypeptide, for example that comprises animmunoglobulin domain); and salt in an amount sufficient to preventaggregation of the polypeptide, thereby stabilizing the composition(e.g., in the absence of arginine or other added amino acid). In someembodiments, compositions provided herein are aqueous compositions(e.g., aqueous solutions). In some embodiments, the polypeptide and saltare provided in water without a buffer. In some embodiments, thecomposition comprises an aqueous buffer or other solvent (e.g., anorganic solvent). In some embodiments, one or more excipients areincluded.

In some embodiments, aspects of the invention relate to arginine-freepolypeptide compositions comprising an isolated polypeptide thatincludes an Fc region of a human immunoglobulin (e.g., IgGl). In someembodiments, aspects of the invention relate to arginine-freepolypeptide compositions comprising an isolated polypeptide thatincludes an extracellular ligand-binding portion of a human p75 tumornecrosis factor (TNF). In some embodiments, aspects of the inventionrelate to arginine-free polypeptide compositions comprising an isolatedpolypeptide that is an extracellular ligand-binding portion of a humanp75 tumor necrosis factor (TNF) receptor fused to the Fc region of ahuman IgGl.

In some aspects, provided herein are compositions comprising (orconsisting, or consisting essentially of): an isolated polypeptide thatis an extracellular ligand-binding portion of a human p75 tumor necrosisfactor receptor fused to the Fc region of a human IgGl; and salt in anamount sufficient to prevent aggregation of the polypeptide, therebystabilizing the composition (e.g., in the absence of arginine or otheradded amino acid). In some embodiments, compositions provided herein areaqueous compositions (e.g., aqueous solutions). In some embodiments, theprotein and salt are provided in water without a buffer. In someembodiments, the composition comprises an aqueous buffer or othersolvent (e.g., an organic solvent). In some embodiments, one or moreexcipients are included.

In other aspects, provided herein are methods comprising combining: anisolated polypeptide that is an extracellular ligand-binding portion ofa human p75 tumor necrosis factor receptor fused to the Fc region of ahuman IgGl; aqueous buffer; and salt in an amount sufficient to preventaggregation of the polypeptide, thereby formulating a stablecomposition.

In yet other aspects, provided herein are methods, comprisingadministering to an individual a composition, comprising: an isolatedpolypeptide that is an extracellular ligand-binding portion of a humanp75 tumor necrosis factor receptor fused to the Fc region of a humanIgGl; aqueous buffer; and salt in an amount sufficient to preventaggregation of the isolated polypeptide, thereby stabilizing thecomposition.

In some embodiments, a composition contains less than 10 mM of freeamino acids (e.g., arginine, lysine and/or glycine). In someembodiments, the composition contains less than 1 mM of free aminoacids. In some embodiments, a composition contains less than 1 mMarginine. In some embodiments, a composition contains less than 0.5 mMarginine. In some embodiments, a composition contains less than 0.1 mM,less than 0.05 mM, less than 0.01 mM, less than 0.005 mM or less than0.001 mM arginine. In some embodiments, the composition does not containfree amino acids. In some embodiments, the composition is substantiallyarginine-free. The isolated polypeptide of any one of the compositionsdescribed herein can comprise, as part of its amino acid sequence,arginine amino acid residues. Arginine residues that, together withother amino acid residues, form the amino acid sequence of a protein arenot considered “free” amino acids. Thus, a composition that “does notcontain amino acids” refers to a composition that does not contain freeamino acids but can contain isolated polypeptide having arginine aminoacid residues as part of is amino acid sequence.

In certain embodiments, compositions described herein comprise about 10mg/ml to about 100 mg/ml of the isolated polypeptide. In someembodiments, the isolated polypeptide is etanercept.

In some embodiments, aqueous buffer is at a concentration of less than100 mM, less than 50 mM, or less than 25 mM. In certain embodiments, theaqueous buffer is at a concentration of about 1 mM to about 15 mM. Insome embodiments, the aqueous buffer is at a concentration of about 1mM. In some embodiments, the aqueous buffer is at a concentration ofless than 1 mM, less than 0.5 mM, less than 0.25 mM, less than 0.1 mM,less than 0.05 mM, or less than 0.01 mM. In some embodiments, theaqueous buffer is sodium phosphate, histidine, potassium phosphate,sodium or potassium citrate, maleic acid, ammonium acetate,tris-(hydroxymethyl)-aminomethane (tris), acetate, diethanolamine or acombination thereof. However, other buffers may be used (e.g., in lowamounts) as aspects of the invention are not limited in this respect).In some embodiments, the compositions described herein do not contain anaqueous buffer. In such embodiments, the proteins in the composition areself-buffering, for example, moderately concentrated proteins can beself-buffering (e.g., in an aqueous solution without added buffer).

In some embodiments, salt is present at a concentration above 50 mM, orabove 100 mM. In some embodiments, the salt is present at aconcentration of about 120 mM to about 150 mM. In some embodiments, thesalt is present at a concentration of greater than 150 mM, depending onthe amount of aqueous buffer present in the solution. Generally, if theamount of aqueous buffer is reduced in the composition, the amount ofsalt (e.g., NaCl) is increased in order to preserve tonicity and thermalstability of the composition. For example, if aqueous buffer is presentin a composition at a concentration of less than 15 mM, then the saltcan be present in the composition at a concentration greater than 150mM. In some embodiments, the salt is sodium chloride. The salt componentof a composition refers to salt in addition to the salt present inaqueous buffer.

In other embodiments, any one of the compositions described herein cancomprise an excipient. The excipient may be sucrose, lactose, glycerol,xylitol, sorbitol, Mannitol, maltose, inositol, trehalose, glucose,bovine serum albumin (BSA), human SA or recombinant HA, dextran, PVA,hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin,polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), polyethyleneglycol, ethylene glycol, glycerol, dimethysulfoxide (DMSO),dimethylformamide (DMF), proline, L-serine, sodium glutamic acid,alanine, glycine, lysine hydrochloride, sarcosine, gamma-aminobutyricacid, TWEEN®-20 (polysorbate 20), TWEEN®-80 (polysorbate 80), SDS,polysorbate, polyoxyethylene copolymer, potassium phosphate, sodiumacetate, ammonium sulfate, magnesium sulfate, sodium sulfate,trimethylamine N-oxide, betaine, zinc ions, copper ions, calcium ions,manganese ions, magnesium ions, CHAPS, sucrose monolaurate,2-O-beta-mannoglycerate or a combination thereof. Other excipients canbe used, as aspects of the invention are not limited in this regard. Inparticular embodiments, the excipient is sucrose. In such embodiments,the sucrose may be at a concentration of from about 0.5% to about 1.5%.In certain embodiments, any of the compositions described herein mayhave a sucrose at a concentration of about 1% by weight.

In some embodiments, any of the compositions described herein can have apH of about 5.5 to about 7.8. In some embodiments, any of thecompositions described herein can have a pH of about 5.8 to about 6.5.In some embodiments, a composition described herein can have a pH of 5.8to 6.5. In some embodiments, a composition can have a pH of 5.8, 5.9,6.0, 6.1, 6.2, 6.3, 6.4, or 6.5.

In one embodiment, the composition comprises (or consists of, orconsists essentially of) 50 mg/ml etanercept, about 10 mM sodiumphosphate, about 140 mM sodium chloride, and about 1% sucrose, whereinthe pH of the composition is about pH 6.0 to about pH 7.0.

In each of the embodiments described herein, the composition is free ofadditional L-arginine (arginine-free). That is, L-arginine is not addedto or combined with the polypeptide in any of the compositions describedherein. The polypeptide itself, however, can contain arginine amino acidresidues, as described elsewhere herein.

Any of the compositions described herein may have a commercially-viableshelf life of at least 24 months.

Any of the compositions described herein may also be suitable forsubcutaneous administration (e.g., non-toxic, purified, sterilized,and/or appropriate isotonicity).

In addition, in any of the compositions described herein, the isolatedpolypeptide may be purified.

In certain embodiments, compositions described herein may be sterilized.

Any of the compositions described herein may be used to treat rheumatoidarthritis, psoriatic arthritis, ankylosing spondylitis, Wegener'sdisease (granulomatosis), Crohn's disease (or inflammatory boweldisease), chronic obstructive pulmonary disease (COPD), Hepatitis C,endometriosis, asthma, cachexia, psoriasis, or atopic dermatitis, orother inflammatory or autoimmune-related illness, disorder, orcondition. The compositions may be administered in an amount sufficientto treat (alleviate symptoms, halt or slow progression of) the disorder(e.g., a therapeutically effective amount).

DETAILED DESCRIPTION OF THE INVENTION

A commercially available soluble form of the TNF receptor fused to an Fcdomain (TNFR:Fc) is known as etanercept. Etanercept (trade name ENBREL®)interferes with tumor necrosis factor (TNF) by acting as a TNFinhibitor. This dimeric fusion polypeptide consisting of theextracellular ligand-binding portion of the human 75 kilodalton (p75)tumor necrosis factor receptor (TNFR) linked to the Fc portion of humanIgGl is currently formulated with L-arginine to prevent aggregation ofthe polypeptide (See U.S. Pat. Nos. 5,447,851 and 7,648,702,incorporated herein by reference).

Arginine, while tolerated by most people, can cause serious side effectsin some people. A severe allergic reaction, called anaphylaxis, canoccur after arginine injections, as well as stomach discomfort,including nausea, stomach cramps, or an increased number of stools.Other potential side effects include low blood pressure and changes innumerous chemicals and electrolytes in the blood, such as highpotassium, high chloride, low sodium, low phosphate, high blood ureanitrogen, and high creatinine levels. In theory, arginine may increasethe risk of bleeding, increase blood sugar levels, increase potassiumlevels, and may worsen symptoms of sickle cell disease. Accordingly,individuals with liver or kidney disease, or those using coagulants arecautioned against using arginine.

As discussed in U.S. Pat. No. 6,748,702, aqueous compositions comprisingFc domain containing polypeptides are thought to require L-arginine inconcentrations of about 1 mM to about 100 mM to prevent aggregation ofthe polypeptides. It is also believed to be necessary for long-termstorage (e.g., two years or more) of such aqueous compositions.

Surprisingly, stable aqueous compositions (e.g., pharmaceuticalcompositions) that are substantially free of L-arginine (e.g., do notcontain a substantial amount of L-arginine) can be prepared such thatthey are stable for a period of two years or more. Applicants have foundthat by increasing the concentration of salt and by decreasing thebuffering capacity of the composition, it is still possible to provide astable polypeptide composition, which can be administered subcutaneouslyto an individual. The term “stable” with respect to long-term storage isunderstood to mean that the active polypeptide of the pharmaceuticalcomposition does not lose more than 20%, more than 15%, more than 10%,or more than 5% of its activity relative to activity of the compositionat the beginning of storage.

In some embodiments, provided herein are compositions, comprising: anisolated polypeptide that is an extracellular ligand-binding portion ofa human p75 tumor necrosis factor receptor fused to the Fc region of ahuman IgGl; aqueous buffer; and salt in an amount sufficient to preventaggregation of the polypeptide, thereby stabilizing the composition,wherein the compositions comprise less than 1 mM concentrations ofL-arginine. In certain other embodiments, the Fc containing polypeptidecompositions are free or substantially free of L-arginine.“Substantially free,” as used herein, refers to a composition withoutadditional free amino acids, such as arginine. It is to be understoodthat the polypeptide itself may comprise the amino acid arginine in itsstructure. In some embodiments, a composition does not contain freearginine amino acids.

As used herein, the phrase “composition” or “compositions” may refer toa formulation(s) comprising a polypeptide prepared such that it issuitable for injection and/or administration into an individual in needthereof. A “composition” may also be referred to as a “pharmaceuticalcomposition.” In certain embodiments, the compositions provided hereinare substantially sterile and do not contain any agents that are undulytoxic or infectious to the recipient. Further, as used herein, asolution or aqueous composition may mean a fluid (liquid) preparationthat contains one or more chemical substances dissolved in a suitablesolvent (e.g., water and/or other solvent, e.g., organic solvent) ormixture of mutually miscible solvents.

In addition, as used herein, the term “about” may mean that there can bevariation in the concentration of a component of the describedcompositions that can be to 5%, 10%, 15% or up to and including 20% ofthe given value. For example, if a composition has about 10 mg/ml of anFc domain containing polypeptide, that composition can have between 8 to12 mg/ml of the stated polypeptide. In certain embodiments, thecompositions comprise about 10 mg/ml to about 100 mg/ml of thepolypeptide. In related embodiments, the compositions comprise 50 mg/mlor about 50 mg/ml of the polypeptide. Compositions may include more orless polypeptide as aspects of the invention are not limited in thisrespect.

In particular embodiments the Fc domain containing polypeptide is asoluble form of the TNF receptor fused to an Fc domain (TNFR:Fc). Acommercially available TNFR:Fc is known as etanercept (ENBREL®, ImmunexCorporation), which is a dimeric fusion polypeptide consisting of theextracellular ligand-binding portion of the human 75 kilodalton (p75)tumor necrosis factor receptor (TNFR) linked to the Fc portion of humanIgGl. The Fc component of etanercept contains the constant heavy 2 (CH2)domain, the constant heavy 3 (CH3) domain and hinge region, but not theconstant heavy 1 (CH1) domain of human IgGl. In some embodiments, an Fcdomain can contain one of the domains described above, while in otherembodiments, an Fc domain can contain all of the domains describedabove. Etanercept is produced by recombinant DNA technology in a Chinesehamster ovary (CHO) mammalian cell expression system. It consists of 934amino acids and has an apparent molecular weight of /approximately 150kilodaltons (Physicians' Desk Reference, 2002, Medical Economics CompanyInc.).

Other polypeptides contemplated for use in particular compositions andmethods described herein include without limitation recombinant fusionpolypeptides comprising at least a portion of an Fc domain of anantibody. A polypeptide fused to an Fc domain and identical to orsubstantially similar to one of the following polypeptides is suitablefor use in the present composition: a flt3 ligand, a CD40 ligand,erythropoietin, thrombopoeitin, calcitonin, Fas ligand, ligand forreceptor activator of NF-kappa B (RANKL), tumor necrosis factor(TNF)-related apoptosis-inducing ligand (TRAIL), thymic stroma-derivedlymphopoietin, granulocyte colony stimulating factor,granulocyte-macrophage colony stimulating factor, mast cell growthfactor, stem cell growth factor, epidermal growth factor, RANTES, growthhormone, insulin, insulinotropin, insulin-like growth factors,parathyroid hormone, interferons, nerve growth factors, glucagon,interleukins 1 through 18, colony stimulating factors, lymphotoxin-β,tumor necrosis factor (TNF), leukemia inhibitory factor, oncostatin-M,and various ligands for cell surface molecules ELK and Hek (such as theligands for eph-related kinases or LERKS).

In certain embodiments, the polypeptides include without limitationrecombinant fusion polypeptides comprising an Fc domain of an antibodyplus a receptor for any of the above-mentioned polypeptides orpolypeptides substantially similar to such receptors. These receptorsinclude without limitation: both forms of TNFR (referred to as p55 andp75), Interleukin-1 receptors (type 1 and 2), Interleukin-4 receptor,Interleukin-15 receptor, Interleukin-17 receptor, Interleukin-18receptor, granulocyte-macrophage colony stimulating factor receptor,granulocyte colony stimulating factor receptor, receptors foroncostatin-M and leukemia inhibitory factor, receptor activator ofNF-kappa B (RANK), receptors for TRAIL (TRAIL receptors 1, 2, 3, and 4),and receptors that comprise death domains, such as Fas orApoptosis-Inducing Receptor (AIR).

In other embodiments, the polypeptides include without limitationdifferentiation antigens (referred to as CD polypeptides) or theirligands or polypeptides substantially similar to either of these, whichare fused to an Fc domain of an antibody. Such antigens are disclosed inLeukocyte Typing VI (Proceedings of the VI^(th) International Workshopand Conference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996).Similar CD polypeptides are disclosed in subsequent workshops. Examplesof such antigens include CD27, CD30, CD39, CD40, and ligands thereto(CD27 ligand, CD30 ligand, etc.). Several of the CD antigens are membersof the TNF receptor family, which also includes 41BB ligand and OX40.The ligands are often members of the TNF family, as are 41BB ligand andOX40 ligand. Accordingly, members of the TNF and TNFR families can beformulated as described herein.

In certain embodiments, enzymatically active polypeptides or theirligands may be used in the compositions and methods described herein.Examples include without limitation recombinant fusion polypeptidescomprising an Fc domain of an antibody fused to all or part of one ofthe following polypeptides or their ligands or a polypeptidesubstantially similar to one of these: metalloproteinase-disintegrinfamily members, various kinases, glucocerebrosidase, superoxidedismutase, tissue plasminogen activator, Factor VIII, Factor IX,apolipoprotein E, apolipoprotein A-I, globins, an IL-2 antagonist,alpha-1 antitrypsin, TNF-alpha Converting Enzyme, ligands for any of theabove-mentioned enzymes, and numerous other enzymes and their ligands.

In some embodiments, the compositions and methods described herein areused to prepare compositions comprising antibodies, human antibodies,humanized antibodies, chimeric antibodies, e.g., antibodies having humanconstant antibody immunoglobulin domains coupled to one or more murinevariable antibody immunoglobulin domain, and/or non-human antibodies, orfragments thereof. Specific examples of antibodies suitable for use inthe present compositions include without limitation commerciallyavailable antibodies such as muromonab-CD3 (Orthoclone OKT-3®, OrthoBiotech), abciximab (REOPRO®, Lilly), rituximab (RITUXAN®, IDEC),dacliximab (ZENAPAX®, Roche Laboratories), basiliximab (SIMULECT®,Novartis), infliximab (REMICADE®, Centocor), palivizumab (SYNAGIS®,MedImmune), trastuzumab (HERCEPTIN®, Genentech), gemtuzuman ozogamicin(MYLOTARG™, Wyeth-Ayerst), and alemtuzumab (CAMPATH®, Berlex). Currentlyeach of the foregoing is available either as a lyophilized powderrequiring rehydration or as a concentrate requiring dilution prior toadministration. The present composition obviates the need for anymanipulations prior to administration, e.g., rehydrating or dilution,while preserving stability of the active ingredients over long-termstorage.

In particular embodiments, the compositions described herein are used tostore polypeptides comprising an antibody conjugated to a cytotoxic orluminescent substance. Such substances include without limitation:maytansine derivatives (such as DM1); enterotoxins (such as aStaphylococcal enterotoxins); iodine isotopes (such as iodine-125);technetium isotopes (such as Tc-99m); cyanine fluorochromes (such asCy5.5.18); and ribosome-inactivating polypeptides (such as bouganin,gelonin, or saporin-S6).

Examples of antibodies or antibody/cytotoxin or antibody/luminophoreconjugates contemplated for use herein include without limitation thosethat recognize one or more of the following antigens: CD2, CD3, CD4,CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52,CD80 (B7.1), CD86 (B7.2), CD147, IL-4, IL-5, IL-8, IL-10, IL-2 receptor,IL-4 receptor, IL-6 receptor, IL-13 receptor, PDGF-β, VEGF, TGF, TGF-β2,TGF-β1, EGF receptor, VEGF receptor, C5 complement, IgE, tumor antigenCA125, tumor antigen MUC1, PEM antigen, LCG (which is a gene productthat is expressed in association with lung cancer), HER-2, atumor-associated glycoprotein TAG-72, the SK-1 antigen, tumor-associatedepitopes that are present in elevated levels in the sera of individualswith colon and/or pancreatic cancer, cancer-associated epitopes orpolypeptides expressed on breast, colon, squamous cell, prostate,pancreatic, lung, and/or kidney cancer cells and/or on melanoma, glioma,or neuroblastoma cells, TRAIL receptors 1, 2, 3 and 4, the necrotic coreof a tumor, integrin alpha 4 beta 7, the integrin VLA-4, B2 integrins,TNF-α, the adhesion molecule VAP-1, epithelial cell adhesion molecule(EpCAM), intercellular adhesion molecule-3 (ICAM-3), leukointegrinadhesin, the platelet glycoprotein gp IIb/IIIa, cardiac myosin heavychain, parathyroid hormone, rNAPc2 (which is an inhibitor of factorVIIa-tissue factor), MHC I, carcinoembryonic antigen (CEA),alpha-fetoprotein (AFP), tumor necrosis factor (TNF), CTLA-4 (which is acytotoxic T lymphocyte-associated antigen), Fc-γ-1 receptor, HLA-DR 10beta, HLA-DR antigen, L-selectin, IFN-γ, Respiratory Syncitial Virus,human immunodeficiency virus (HIV), hepatitis B virus (HBV),Streptococcus mutans, and Staphylococcus aureus.

In some embodiments, the compositions described herein are used foranti-idiotypic antibodies, or substantially similar polypeptides,including without limitation anti-idiotypic antibodies against: anantibody targeted to the tumor antigen gp72; an antibody against theganglioside GD3; or an antibody against the ganglioside GD2.

In other embodiments, the Fc domain containing polypeptide used in thecompositions described herein are produced by living host cells thatexpress the polypeptide, such as hybridomas in the case of antibodies,or host cells that that have been genetically engineered to produce thepolypeptide in the case of fusion polypeptides or antibodies. Methods ofgenetically engineering cells to produce polypeptides are well known inthe art. See, e.g., Ausubel et al., eds. (1990), Current Protocols inMolecular Biology (Wiley, New York). Such methods include introducingnucleic acids that encode and allow expression of the polypeptide intoliving host cells. These host cells can be without limitation bacterialcells, fungal cells, or animal cells grown in culture. Bacterial hostcells include without limitation Escherichia coli cells. Examples ofsuitable E. coli strains include without limitation: HB101, DH5α,GM2929, JM109, KW251, NM538, NM539, and any E. coli strain that fails tocleave foreign DNA. Fungal host cells that can be used include withoutlimitation Saccharomyces cerevisiae, Pichia pastoris and Aspergilluscells. A few examples of animal cell lines that can be used are CHO,VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, and W138. New animal cell linescan be established using methods well know. by those skilled in the art(e.g., by transformation, viral infection, and/or selection).Optionally, the polypeptide can be secreted by the host cells into themedium.

In certain embodiments, the expressed Fc domain containing polypeptideare purified by any standard method. When the Fc domain containingpolypeptide is produced intracellularly, the particulate debris isremoved, for example, by centrifugation or ultrafiltration. When thepolypeptide is secreted into the medium, supernatants from suchexpression systems can be first concentrated using standard polypeptideconcentration filters. Protease inhibitors can also be added to inhibitproteolysis and antibiotics can be included to prevent the growth ofmicroorganisms.

In some embodiments, the Fc domain containing polypeptide are purifiedusing, for example, hydroxyapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, and/or any combination ofpurification techniques known or yet to discovered. For example, proteinA can be used to purify Fc domain containing polypeptides that are basedon human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al.,1983, J. Immunol. Meth. 62:1-13). Protein G is recommended for all mouseisotypes and for human gamma 3 (Guss et al., 1986, EMBO J. 5:1567-1575).

Other techniques for polypeptide purification such as fractionation onan ion-exchange column, ethanol precipitation, reverse phase HPLC,chromatography on silica, chromatography on heparin SEPHAROSE™ (acrosslinked, beaded-form of agarose), chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation can alsobe utilized depending on need. Other polypeptide purificationtechniques/methods can be used.

In particular embodiments, the compositions described herein areprepared by combining, in addition to a purified polypeptide describedabove, buffer, salt (e.g., NaCl), and an additional excipient (e.g.,sucrose). In some embodiments, the present compositions comprise lessthan 1 mM L-arginine, while in other embodiments, the compositionsdescribed herein are free or substantially free of arginine (e.g.,L-arginine). It will be understood one of ordinary skill in the art thatthe combining of the various components to be included in thecomposition can be done in any appropriate order, namely, the buffer canbe added first, middle or last and the tonicity modifier can also beadded first, middle or last. It is also to be understood by one ofordinary skill in the art that some of these chemicals can beincompatible in certain combinations, and accordingly, are easilysubstituted with different chemicals that have similar properties butare compatible in the relevant mixture.

Aggregation inhibitors reduce a polypeptide's tendency to associate ininappropriate or unwanted ternary or quaternary complexes. Surprisingly,the present inventors have found that by increasing the salt and bydecreasing the buffer capacity in a composition comprising an Fccontaining polypeptide, there is no need for the addition of free aminoacids (e.g., arginine, lysine, glycine). The polypeptides within thearginine-free compositions remain active (effective) and can be storedfor at least 24 months. In certain embodiments, the salt concentrationis greater than 100 mM, while in other embodiments, the saltconcentration is about 140 mM, or greater. Salts, used herein, caninclude without limitation sodium chloride (NaCl), potassium chloride(KCl), sodium citrate (Na₃C₆H₅O₇.2H₂O), magnesium sulphate (MGSO₄),calcium chloride (CaCl), sodium hypochlorite (NaClO), sodium nitrate(NaNO₃), mercury sulphide (HgS), sodium chromate (Na₂CrO₄) and magnesiumdioxide (MgO₂). Salt both maintains the isotonicity and the thermalstability of the composintion, for example, in the absence of arginine(e.g., L-arginine).

Buffering agents maintain pH in a desired range and various bufferssuitable for use in the compositions described herein include withoutlimitation histidine, potassium phosphate, sodium or potassium citrate,maleic acid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris),various forms of acetate and diethanolamine. In certain embodiments, thebuffer is sodium phosphate as its buffering capacity is at or near pH6.2. In some embodiments, the concentration of the buffer in thecompositions is about 25 mM, or less. In some embodiments, theconcentration of the buffer is 25 mM. In particular embodiments, theconcentration of the buffer is about 10 mM, or less. In someembodiments, the concentration of the buffer is 10 mM. Buffers are wellknown in the art and are manufactured by known methods and availablefrom commercial suppliers.

When the pH of the composition is set at or near physiological levels,comfort of the individual upon administration is maximized. In certainembodiments, the pH is about 5.8 to 8.4. In other embodiments, the pH iswith about 6.2 to 7.4. It is to be understood that the pH can beadjusted as necessary to maximize stability and solubility of thepolypeptide in a particular composition and as such, a pH outside ofphysiological ranges, yet tolerable to the individual, is within thescope of the invention.

In certain embodiments, excipients, also referred to as chemicaladditives, co-solutes, or co-solvents, that stabilize the polypeptidewhile in solution (also in dried or frozen forms) are added to acomposition. Examples include but are not limited to sugars/polyols suchas: sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose,inositol, trehalose, glucose; polymers such as: serum albumin (bovineserum albumin (BSA), human SA or recombinant HA), dextran, PVA,hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin,polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC); non-aqueoussolvents such as: polyhydric alcohols, (e.g., PEG, ethylene glycol andglycerol) dimethysulfoxide (DMSO) and dimethylformamide (DMF); aminoacids such as: proline, L-serine, sodium glutamic acid, alanine,glycine, lysine hydrochloride, sarcosine and gamma-aminobutyric acid;surfactants such as: TWEEN-80™ (polysorbate 80), TWEEN-20™ (polysorbate20), SDS, polysorbate, polyoxyethylene copolymer; and miscellaneousexcipients such as: potassium phosphate, sodium acetate, ammoniumsulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide,betaine, metal ions (e.g., zinc, copper, calcium, manganese, andmagnesium), CHAPS, monolaurate, 2-O-beta-mannoglycerate or anycombination of the above.

In certain embodiments, the concentration of one or more excipients in acomposition described herein is/are about 0.001 to 5 weight percent,while in other embodiments, the concentration of one or more excipientsis/are about 0.1 to 2 weight percent. Excipients are well known in theart and are manufactured by known methods and available from commercialsuppliers. In some embodiments, the excipient is sucrose. In otherembodiments, sucrose is present in the composition at a concentration ofabout 1 percent.

In a particular embodiment, a composition described herein comprises (orconsists of, or consists essentially of) about 25 to about 50 mg TNFR:Fc(e.g., etanercept), about 10 mM to about 50 mM sodium phosphate (e.g.,monobasic and/or dibasic), about 0.75% to about 1.25% sucrose, about 50mM to about 150 mM NaCl, at about pH 6.0 to about pH 7.0.

In another embodiment, a composition described herein comprises (orconsists of, or consists essentially of) about 50 mg/ml TNFR:Fc, about10 mM sodium phosphate, about 140 mM sodium chloride, and about 1%sucrose at about pH 6.2.

In certain embodiments, provided herein are methods of treating anindividual comprising administering to the individual a therapeuticallyeffective amount of the composition described herein, wherein theindividual has a disease or disorder that can be beneficially treatedwith a Fc domain containing polypeptide in the composition. In someembodiments, the Fc domain containing polypeptide is derived from thesame species of individual as is to be treated with the composition. Inparticular embodiments, the individual is a human in need of treatment.When the Fc domain containing polypeptide of the composition is TNFR:Fc,examples of diseases or disorders that can be treated include but arenot limited to rheumatoid arthritis, psoriatic arthritis, ankylosingspondylitis, Wegener's disease (granulomatosis), Crohn's disease (orinflammatory bowel disease), chronic obstructive pulmonary disease(COPD), Hepatitis C, endometriosis, asthma, cachexia, psoriasis, andatopic dermatitis. Additional diseases or disorders that can be treatedwith TNFR:Fc include those described in WO 00/62790, WO 01/62272 andU.S. Patent Application No. 2001/0021380.

In other aspects, provided herein are polypeptide compositions havingimproved long-term storage such that the active ingredient, e.g., an Fcdomain containing polypeptide, is stable over the course of storage inliquid (or frozen) states. As used herein, the phrase “long-term”storage is understood to mean that the composition can be stored forthree months or more, for six months or more, or for one year, or twoyears, or more. Long term storage is also understood to mean that thecomposition is stored either as a liquid at 2-8° C. or is frozen, e.g.,at −20° C. or colder. In certain embodiments, the composition can befrozen and thawed more than once. The term “stable” with respect tolong-term storage is understood to mean that the active polypeptide ofthe composition does not lose more than 20%, or 15%, or even 10% of itsactivity. In particular embodiments, the active polypeptide of thecomposition does not lose more than 5% of its activity relative toactivity of the composition at the beginning of storage. Stability of acomposition can be assessed based on potency, appearance, concentration,pH, and oxidation, and can be assessed using, for example, hydrophobicinteraction chromatography (HIC), capillary electrophoresis-sodiumdodecyl sulfate (CE-SDS), high accuracy (HIAC) liquid particle counters,and/or isoelectric focusing. Other protein stability assays are known inthe art and can be used herein.

The appropriate dosage, or therapeutically effective amount, of the Fcdomain-containing polypeptide of the compositions will depend on thecondition to be treated, the severity of the condition, prior therapy,and the individual's clinical history and response to the therapeuticagent. The proper dose can be adjusted according to the judgment of theattending physician such that it can be administered to the individualone time or over a series of administrations. The composition can beadministered as a sole therapeutic or in combination with additionaltherapies as needed.

In certain embodiments, the effective Fc domain containing polypeptideamount per adult dose ranges from about 1-500 mg/m², or from about 1-200mg/m², or from about 1-40 mg/m² or about 5-25 mg/m². Alternatively, aflat dose may be administered, whose amount may range from 2-500mg/dose, 2-100 mg/dose or from about 10-80 mg/dose. If the dose is to beadministered more than one time per week, an exemplary dose range is thesame as the foregoing described dose ranges or lower and preferablyadministered two or more times per week at a per dose range of 25-100mg/dose. In other embodiments, an acceptable dose for administration byinjection contains 80-100 mg/dose, or alternatively, contains 80mg/dose. The dose can be administered at biweekly, weekly doses, orseparated by several weeks (for example 2 to 8). In a particularembodiment, TNFR:Fc (etanercept) is administered at 25 mg by a singlesubcutaneous (SC) injection. Other routes of administration arecontemplated.

In many instances, an improvement in an individual's condition will beobtained by a dose of up to about 100 mg of the composition one to threetimes per week over a period of at least three weeks, though treatmentfor longer periods may be necessary to induce the desired degree ofimprovement. For incurable chronic conditions the regimen may becontinued indefinitely. For pediatric individuals (ages 4-17), asuitable regimen involves a dose of 0.4 mg/kg to 5 mg/kg of a thepolypeptides of the invention, administered one or more times per week.

In other embodiments, the compositions described herein are prepared ina bulk formulation and as such, the components of the composition areadjusted so that it is higher than would be required for administrationand diluted appropriately prior to administration.

In certain embodiments, the compositions described herein areadministered parenterally, e.g., subcutaneously, intramuscularly,intravenously, intraperitoneal, intracerebrospinal, intra-articular,intrasynovial, and/or intrathecal. Parenteral administration can be bybolus injection or continuous infusion. compositions for injection maybe presented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. In addition, a number of recentdrug delivery approaches have been developed and the compositions of thepresent invention are suitable for administration using these newmethods, e.g., INJECT-EASE™ (automatic injector), GENJECT™ (injectiondevice), injector pens such as GENPEN™, and needleless devices such asMEDIJECTOR™ and BIOJECTOR™. The present composition can also be adaptedfor yet to be discovered administration methods. See also Langer, 1990,Science, 249:1527-1533.

In some embodiments, the compositions described herein are formulated asa depot preparation. Such long acting compositions may be administeredby implantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the compositions may bemodified with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

In other embodiments, the compositions described herein are presented ina vial, pack or dispenser device which may contain one or more unitdosage forms containing the active ingredient. In some embodiments, thedispenser device comprises a syringe having a single dose of the liquidcomposition ready for injection. The syringe can be accompanied byinstructions for administration.

In other aspects, provided herein are kits or containers, which containan aqueous composition of the invention. The concentration of thepolypeptide in the aqueous composition can vary over a wide range. Incertain embodiments, it ranges of from about 0.05 to about 20,000micrograms per milliliter (μg/ml) of aqueous composition. The kit canalso be accompanied by instructions for use.

The compositions are further described below by way of non-limitingexamples.

EXAMPLES Example 1 One Embodiment of a Stable Polypeptide Composition

TABLE 1 Etanercept composition. Amount per mL of Ingredient Function thedosage form Grade Etanercept Active 50 mg N/A ingredient Sodium Buffer0.67 mg  10 mM USP phosphate, dibasic, heptahydrate Sodium Buffer 1.04mg USP phosphate, monobasic, monohydrate Sodium chloride Stabilizer 8.18mg 140 mM USP/Ph. Eur. (NaCl) Sucrose Stabilizer 0.01 mg 1% NF/Ph. Eur.Water Solvent Q.S. USP/Ph. Eur. (for injection)

Example 2 Stability Data for the Polypeptide Composition at −70° C.

TABLE 2 Stability Data for Etanercept at −70° C. Expected Time Points(Months) Test Method Result/Range Initial 1 3 6 Appearance Color < Y3,Y7-Y6 Y7-Y6 Y7-Y6 Y7-Y6 Clarity < 80NTU, 3-6NTU 0-3NTU 3-6NTU 3-6NTUReport (FIO) visible particles Protein 45.0 to 55.0 48.2 49.4 51.5 51.0Concentration mg/mL By UV Size Exclusion Report % HMW, 1.2 1.1 1.1 1.2Chromatog- expected result raphy (SEC) is ≤5.0% HMW

Example 3 Stability Data for the Polypeptide Composition at 5±3° C.

TABLE 3 Stability Data for Etanercept at 5 ± 3° C. Expected Time Points(Months) Test Method Result/Range Initial 1 3 6 Appearance Color < Y3,W-Y7 Y7-Y6 Y7-Y6 Y7-Y6 Clarity < 80NTU, 3- W- 6- 3- Report (FIO) 6NTU3NTU 18NTU 6NTU visible particles Particle Particle Particle ParticleProtein Conc. 45.0 to 55.0 47.8 49.3 51.3 50.0 By UV mg/mL SizeExclusion Report % HMW, 1.2 1.3 1.3 1.6 Chromatog- expected result raphy(SEC) is ≤5.0% HMW Participate Report 460 435 135 180 result ≥10 μmReport 110 25 55 25 result ≥25 μm

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present invention is notintended to be limited to the above Description, but rather is as setforth in the appended claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process. Furthermore, it is to be understood that theinvention encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the listed claims is introduced into anotherclaim. For example, any claim that is dependent on another claim can bemodified to include one or more limitations found in any other claimthat is dependent on the same base claim.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It shouldit be understood that, in general, where the invention, or aspects ofthe invention, is/are referred to as comprising particular elements,features, etc., certain embodiments of the invention or aspects of theinvention consist, or consist essentially of, such elements, features,etc. For purposes of simplicity those embodiments have not beenspecifically set forth in haec verba herein. It is also noted that theterm “comprising” is intended to be open and permits the inclusion ofadditional elements or steps.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or sub-rangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

The term “about” or “approximately” can mean within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, e.g., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviations,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, up to 10%, up to 5%, or up to 1% of a given value.Alternatively, the term can mean within an order of magnitude, forexample, within 5-fold, or within 2-fold, of a value. Where particularvalues are described in the application and claims, unless otherwisestated the term “about” meaning within an acceptable error range for theparticular value should be assumed.

In addition, it is to be understood that any particular embodiment ofthe present invention that falls within the prior art may be explicitlyexcluded from any one or more of the claims. Since such embodiments aredeemed to be known to one of ordinary skill in the art, they may beexcluded even if the exclusion is not set forth explicitly herein. Anyparticular embodiment of the methods of the invention can be excludedfrom any one or more claims, for any reason, whether or not related tothe existence of prior art.

What is claimed is:
 1. A composition, comprising: an isolated polypeptide that is an extracellular ligand-binding portion of a human p75 tumor necrosis factor receptor fused to the Fc region of a human IgGl at a concentration of 10 mg/mL to 100 mg/mL; aqueous buffer at a concentration of less than 25 mM, wherein the aqueous buffer is sodium phosphate, potassium phosphate, sodium or potassium citrate, maleic acid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris), acetate, diethanolamine, or a combination thereof; salt at a concentration of greater than 100 mM; and a sugar, wherein the composition does not contain free amino acids, and wherein the composition is suitable for injection.
 2. The composition of claim 1, wherein the isolated polypeptide is etanercept.
 3. The composition of claim 1, wherein the aqueous buffer is present at a concentration of 1 mM to 15 mM.
 4. The composition of claim 1, wherein the salt is sodium chloride.
 5. The composition of claim 1, wherein the salt is present at a concentration of 120 mM to 150 mM.
 6. The composition of claim 1, wherein the sugar is sucrose, lactose, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose, or a combination thereof.
 7. The composition of claim 6, wherein the sugar is sucrose.
 8. The composition of claim 1, wherein the pH of the composition is 5.5 to 7.8.
 9. A method comprising administering to an individual the composition of claim
 1. 10. A composition, comprising: an isolated polypeptide that is an extracellular ligand-binding portion of a human p75 tumor necrosis factor receptor fused to the Fc region of a human IgGl at a concentration of 10 mg/mL to 100 mg/mL; aqueous buffer at a concentration of less than 25 mM, wherein the aqueous buffer is sodium phosphate, potassium phosphate, sodium or potassium citrate, maleic acid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris), acetate, diethanolamine, or a combination thereof; salt at a concentration of greater than 100 mM; and sucrose at a concentration of from 0.5% to 1.5%, wherein the composition does not contain free amino acids, and wherein the composition is suitable for injection.
 11. The composition of claim 10, comprising 50 mg/ml etanercept, about 10 mM sodium phosphate, 140 mM sodium chloride, and 1% sucrose, wherein the pH of the composition is pH 6.0 to pH 7.0.
 12. A composition, consisting essentially of: an isolated polypeptide that is an extracellular ligand-binding portion of a human p75 tumor necrosis factor receptor fused to the Fc region of a human IgGl; aqueous buffer at a concentration of 10 mM, wherein the aqueous buffer is sodium phosphate, potassium phosphate, sodium or potassium citrate, maleic acid, ammonium acetate, tris-(hydroxymethyl)-aminomethane (tris), acetate, diethanolamine, or a combination thereof; salt at a concentration of 140 mM; and sucrose, wherein the composition does not contain free amino acids, and wherein the composition is suitable for injection.
 13. The composition of claim 12, wherein the polypeptide is etanercept.
 14. The composition of claim 12, wherein the sucrose is at a concentration of 0.5% to 1.5%. 